The long-term objective of this application is to understand the molecular pathogenesis of infection with the intracellular protozoan Toxoplasma gondii. The parasite is an important pathogen in immunosuppressed populations and is classified by the NIAID as a category B biodefense organism. Nevertheless, in most cases infection with this widespread parasite is asymptomatic. Host survival during infection requires robust Type 1 cytokine production, yet overly exuberant responses can result in host pathology and death. Thus, both parasite and host benefit from regulated pro-inflammatory cytokine responses. The underlying hypothesis to be tested is that T. gondii possesses molecular mechanisms to subvert host signaling pathways leading to pro-inflammatory cytokine production. Recent work has revealed the ability of Toxoplasma to down-regulate cytokines such as TNF-1 from within infected cells. Increasing evidence indicates this is achieved through sophisticated manipulation of host intracellular signaling cascades. The goal of this proposal is to elucidate mechanisms by which Toxoplasma subverts pro-inflammatory signaling cascades in the innate immune system, in particular within cells such as macrophages and dendritic cells that are targeted for in vivo infection. Cellular and biochemical methods will be employed to define the points at which Toxoplasma interferes with host signal transduction. In this regard, parasite-induced activation of signaling molecule STAT3, known to be involved in down-regulation of pro-inflammatory cytokines IL-12 and TNF-1, will be examined. The nature of TNF-1 suppression in infected macrophages is profound. Therefore, we will employ molecular methods to determine how the parasite influences promoter activity of the TNF-1 gene. The last part of this proposal will focus effort on examining if parasite-infected macrophages and dendritic cells display a suppression phenotype during in vivo mouse infections. This will be accomplished using cellular and immunological methods to determine IL-12 and TNF-1 production in infected cells in vivo and their in vivo ability to respond to defined Toll-like receptor ligands. In addition, macrophage and dendritic cell expression of cell surface activation markers will be analyzed. From these studies, we expect to gain important information on how T. gondii interacts with the host innate immune system, allowing a deeper understanding of infection with this important opportunistic pathogen. PROJECT NARRATIVE / RELEVANCE: The relevance of the project to public health is that Toxoplasma infects between 30-80% of the human population worldwide. While normally an asymptomatic infection, with suboptimal immune function the parasite emerges as a devastating and sometimes lethal infection. This project will enhance our understanding of immunity to the parasite, leading to improved treatment strategies.